Repetitive DNA sequences in the immunoglobulin switch mu region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5 kb region spanning the intronic enhancer to the constant mu gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)-deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1 kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung-/- and Aid-/-Ung-/- mice, but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung-/- B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, that are incorporated by DNA polymerase eta, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase eta during hypermutation.